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Abstract

The use of cacao, in its familiar food and beverage form, dates back about 3,800 years.
By the time of the discovery of America by Europeans, the Aztecs and Mayans had developed
methods for drying, roasting and grinding cacao beans for use in beverages and foods.
Today, the same processes are used, but have been adapted to modern machinery. The
current study was conducted to compare the traditional Mesoamerican processing and
modern processing methods and their impact on the antioxidant activity and flavan-3-ol
content of the resulting processed cocoa mass. Two cocoa bean types were used: unfermented
cocoa beans commonly produced and consumed today by the native peoples of Mesoamerica,
and fermented Ivory Coast cocoa beans representing the most common country of origin
for cocoa in the world today. Both bean types were processed by roasting and grinding
using traditional and modern processing methods. The traditional method consisted
of roasting whole beans on an earthenware comal heated with wood charcoal, de-shelling
the beans, then grinding the beans to a paste using a stone metate and mano. The modern
method consisted of roasting cocoa beans with pilot-scale equipment that simulates
large-scale chocolate manufacturing using whole bean drum roasters, deshelling the
beans followed by mechanical grinding using a ball mill producing a chocolate paste.
The antioxidant capacity and flavanol content of these chocolate pastes were then
determined. The data show that the antioxidant capacity and flavanol content of unprocessed
Mexican Lavado cocoa beans were significantly higher than that of the unprocessed,
fermented Ivory Coast cocoa beans. In unfermented Mexican Lavado cocoa beans, the
traditional and modern processing revealed no clear advantage with respect to the
antioxidant and flavanol contents. However, for fermented Ivory Coast beans, there
were some differences in the level of flavanols between traditionally processed and
modern processed fermented beans. These differences, while significant for some, but
not all of the flavanol measures, were attributed to final roast temperatures achieved
and to the possible lack of uniformity of bean fermentation.

Keywords:

Introduction

Cacao originated and evolved in the tropical rainforests of the upper Amazon and Orinoco
Rivers in South America [1]. When fresh fruit of cacao, also known as cocoa or cacao pods, are harvested, the
hard woody pod is broken open and the cacao seeds, better known as cocoa beans, with
their surrounding white, sweet pulp, are revealed. Fresh cacao beans at this stage
taste bitter and contain little or no cacao flavor [2-4]. The archeological evidence dating back to about 1900 to 1400 BC, indicates that
cacao juice expressed from the sweet pulp was the first product of the cacao seed
to be used [5]. The archeological records suggest that the first consistent use of the brown, roasted
cacao paste that was made into cocoa drinks, sauces and solid cocoa-containing foods
dates back to about 1,000 BC [6]. The forms of cocoa that we are familiar with today have these brown cocoa solids
—chocolate liquor and cocoa powder—which are the ingredients of chocolate candy, cakes,
cookies and hot cocoa beverages.

While no one really knows how the processing of cocoa beans by fermentation, roasting
and grinding actually developed, Edgar [7] speculates that Mayans routinely fermented cocoa beans for one to two days with their
pulp to produce alcohol. He then speculates that if the Mayans separated the juice
and dried the beans, the beans could be subsequently roasted on a comal found in most
kitchens used to cook maize tortillas and stews (Figure 1) to produce cocoa aroma and beans with less bitterness. Indeed modern science has
learned that fermentation lowers the bitterness of roasted cocoa beans as well as
their content of flavan-3-ols [8,9] while also creating more chocolate flavor when the beans are roasted [4]. Roasting also makes the inedible shells brittle and easy to remove. To make chocolate
paste, Edgar speculates that roasted beans were ground using a kitchen grinding stone
called a metate and a stone mano (Figure 2) which were also commonly available in households to make maize flour. We refer to
this as the Traditional Process. These same processes of roasting, shell removal and
milling have been mechanized and we refer to this as the Modern Process.

Figure 1.Traditional bean roasting. Comal (Left) with bricks to elevate it above charcoal heat from beneath. Bean roasting
(Right) in which 250 gm of Lavado beans are being prepared.

Figure 2.Traditional bean grinding. The metate base with the mano pestle on its surface (left). Bean grinding (right)
on the metate. Note the charcoal heat being applied from beneath of the metate.

Cacao is known to contain very high levels of flavan-3-ols (flavanols) that occur
both as monomers of epicatechin and catechin and as polymerized flavanols, or procyanidins
[9-13]. The flavanols are commonly associated with cardiovascular health benefits which
include lower rates of morbidity and mortality due to stroke and heart attack, improved
vascular endothelial function, reduction in blood pressure and increases in HDL cholesterol
[14,15].

Studies have shown that flavanols are lost during transformation of cocoa beans into
chocolate. Therefore, one can ask the compelling question regarding the level of these
flavanols: Do traditional processing methods for roasting cacao beans have an advantage
over the modern methods used today to produce roasted cocoa beans for making chocolate,
perhaps because the process is more natural or not mechanized? The objective of this
study is to compare the level of antioxidant capacity, flavanol monomers as well as
oligomers and total flavanols in cacao beans processed by traditional Mesoamerican
methods to the levels found in cocoa beans processed by modern methods. Here we have
studied two types of cacao beans: unfermented cacao beans from southern Mexico, locally
known as Lavado beans (lavar, in Spanish, meaning to wash) and conventionally fermented
Ivory Coast beans. Beans of each type then were processed in a modern pilot plant
to simulate industrial processing and were compared to beans prepared by the traditional
Mesoamerican method which included comal roasting, hand winnowing and stone grinding
on a metate.

Material and methods

Cocoa bean samples

Two types of cocoa beans were obtained for this study: Mexican unfermented Lavado
and Ivory Coast fermented. The Mexican Lavado beans were obtained from AMCO (Veracruz,
Mexico) and were processed according to the local Lavado tradition in the Chiapas,
Mexico region. The beans were removed from ripe pods, washed by hand with water to
remove most of the pulp, then sun-dried. This results in unfermented, terracotta colored
beans which are typical from this region of Mexico (Figure 3). Ivory Coast beans were obtained as standard fair average quality beans through
The Hershey Company’s purchasing department. These beans typically are heap-fermented
for four to six days and then sun-dried. Ivorian beans have the typical brown appearance
of fermented beans (Figure 3, bottom, right). The dried Lavado and the Ivorian beans had a moisture content of
between 5.5% to 6.5% and fat content of 51% to 52%.

Traditional processing method

Cocoa beans were roasted using methods that resembled traditional, ancient processing
and typical of those still used today in Central America for local use. A 50-cm diameter
earthenware comal was obtained from a Mexican market and was supported by several
bricks. The comal was heated from beneath with charcoal (Figure 1). Once the center of the comal reached 250–300°C surface temperature as measured
with a RayTek Mini-Temp Model MT6 laser thermometer (Fisher Scientific), 250 g of
whole, dried cocoa beans were placed on the comal and heated. The beans were stirred
occasionally with a spatula to avoid burning the outer shell. When beans developed
a characteristic cacao aroma, they were heated for another three to four minutes,
removed from the comal and cooled. Final external bean temperatures were 125–130°C
as measured by a RayTek laser thermometer. The shells of roasted beans were fractured
into small pieces using a stone mano and removed by hand or winnowed by sifting and
blowing away the lighter shells from the heavier cocoa nibs. Shell-free nibs were
ground using a stone metate and mano purchased from Melissa Guerra Cookware (Figure 2; bottom). The metate was kept slightly warm by charcoal underneath. The nibs were
ground several times to produce a smooth, melted paste with a glossy appearance. Several
batches were processed, combined and three aliquots were taken for analysis.

Modern processing method

Modern industrial roasting was accomplished on a pilot plant scale using 1362 g of
whole, dried cocoa beans in a drum-style US Roaster. Burner temperature was set at
150°C and 2% water was added at the beginning of the roast. The beans were roasted
to a final moisture content of less than 1% with a bean temperature of about 155°C.
Shell-free nibs were obtained using a bench-top BLT cracker and winnower. Nibs were
made into liquor with a bench-top Netsch Ball Mill and ground to a 25 μm particle
size. Several batches were processed, combined, and three aliquots were taken for
analysis.

Sample preparation

Composite samples from each type of roasting were analyzed, to avoid measuring batch
to batch variability but rather focusing on the impact of the different processing
methods. In all cases, unroasted and roasted cocoa beans by both traditional and modern
processing methods had their shells removed and were ground to a consistent particle
size. Prior to analysis for the three flavanol tests described below, the ground mass
was defatted by extracting three times with hexane [16]. Final results are reported on per gram of original cocoa bean material.

Antioxidant capacity measurement

Total antioxidant activity of unroasted and roasted cocoa bean samples was measured
by the ORAC assay. ORAC is a widely used fluorescent method for assessing antioxidant
capacity in food and biological samples. It is based on the inhibition of a peroxy
radical induced oxidation initiated by the thermally based decomposition of 2, 2'-
azobis- 2- methyl- propanimidamide, using fluorescein as a fluorescent probe and Trolox
as a standard substrate. The results are expressed as μmol Trolox equivalent (TE)
per gram [17,18].

Flavanol monomer determination

The flavanol monomers (±)-catechin and (±)-epicatechin were measured by the HPLC method
of Nelson and Sharpless [19] standardized with (+)-catechin hydrate and (−)-epicatechin using the method recently
validated using a five-laboratory ring test [20]. We define and report measures of (±)-epicatechin as epicatechin and (±)-catechin
as catechin.

Flavanol monomer to decamer determination

The flavanol oligomers with degree of polymerization (DP) ranging from DP 1 to 10
were measured by the HPLC method of Robbins et al.[21] with each sample extract standardized using individual purified oligomeric standards
isolated and characterized as described by Hurst et al.[22]. These standards were obtained from Planta Analytica (Danbury, CT). The data are
reported as Flavanols DP 1–10.

Total flavanol determination

Total flavanols were measured by the DMAC method which has been validated by a three
laboratory ring-test described by Payne et al.[23]. This method is based on the specific reaction of p-dimethylaminocinnamaldehyde with the flavanol structure. The test was standardized
using procyanidin B2 purchased from Planta Analytica (Danbury, CT). The method is
specific to monomeric and polymeric flavan-3-ols as well as to related compounds such
as epigallocatechin gallate and epicatechin-3-gallate. Monomers, i.e., epicatechin
and catechin, react more strongly than higher MW oligomers with DMAC. Thus mg/g may
not change, but the various proportions of monomers to DP2 and higher may be altered
with proces sing. These results are reported as Total Flavanols.

Statistical analysis

The data presented in this paper represent analysis of three representative samples
for each sample type. Values for antioxidant activity and flavanol contents are expressed
as averages and standard deviations. Due to the small sample populations, p-values
were calculated on the basis of a significant difference. Therefore, at a 95% confidence
level, p-values ≤0.05 represent a significant difference between two sets of data.

Results and discussion

Comparison of unprocessed Lavado and Ivorian beans

Table 1 provides the antioxidant and flavanol content for the unroasted Lavado and Ivory
Coast cocoa bean material. The current results agree with previously published literature
[9-13], namely that less fermented Lavado nibs have higher ORAC values and more flavanol
content than fermented Ivory Coast nibs as measured by flavanol monomers, flavanol
DP 1–10 and by total flavanols. As the literature has shown, ancient cultures would
most likely have used less-fermented, Lavado-like cocoa beans. In fact, today there
are areas ranging from southern Mexico to Venezuela where Lavado beans are preferred
for local use. The reason is that beans washed free of the surrounding pulp are more
easily dried during the rainy season and less subject to spoilage. However, today,
chocolate manufacturers in the developed world rely on fermented beans like those
from the Ivory Coast, because fermentation creates more chocolate flavor upon roasting
and reduces the bitterness [3,4].

Since the focus of this study was a comparison of processing methods, the data presented
in the following tables and graphs compare only the impact of traditional and modern
bean processing on various antioxidant and flavanol measurements. A comparison will
not be made to the unroasted starting material because the difference in moisture
contents would create an unfair comparison.

Processing of Lavado (unfermented) beans

The data in Table 2 and Figure 4 shows a comparison of the antioxidant capacity and flavanol content of Lavado beans
roasted by the traditional and modern methods. The major and perhaps surprising observation
is that there were no significant differences in antioxidant capacity, epicatechin,
flavanols DP 1 – 10 and total flavanols. The catechin content of the modern processed
beans, however, was significantly higher than traditionally processed beans. The results
in Table 2 and Figure 4 are perhaps surprising since it has been found that increased roast temperatures
can decrease flavanol content when temperatures in excess of 70°C are reached [13]. Since the modern processed beans reached a higher terminal temperature of 150°C
compared to the 125°C to 130°C exterior temperature of the traditionally processed
beans, we might have expected to see lower antioxidant activities and flavanol values
in the modern processed Lavado beans, but this was not observed.

Figure 4.Comparison of unfermented (Lavado) cacao beans that have been left unprocessed, have
been roasted and ground using traditional methods (Traditional Process) and have been
processed using modern processing. Top Graph. Analysis of antioxidant activity (ORAC); Second Graph. Analysis of epicatechin
(yellow bars) and catechin (blue bars) content; Third Graph, Analysis of Flavanols
DP 1–10 content and Bottom Graph, Analysis of Total Flavanol content.

Table 2.Antioxidant capacity and flavanol content comparison for unfermented Lavado cocoa
beans roasted on a Mayan-style comal and ground on a metate (Traditional process)
compared to the same beans roasted and ground using modern pilot plant equipment (Modern
process)

Our results with unfermented beans are different from those of Cervelotti et al. in 2008 [24] who comparing the in vitro antioxidant activity and polyphenol content of finished chocolates made for standard,
large scale manufactured products and for artisan manufacturing methods which used
small batch roasting and stone, melangeur grinding. They concluded that artisan-made
chocolate had higher antioxidant capacities when compared to mass produced chocolate
as measured by Briggs-Rauscher reaction and by the Trolox Equivalent Antioxidant Capacity
(TEAC) methods. They also found higher levels of polyphenols in the artisan chocolates.
But their study made no attempt to measure the flavan-3-ol content of the chocolates,
nor were they able to measure the antioxidant activities or the total polyphenol composition
of the starting materials, as we have. Additionally, the Cervelotti study did not
attempt to measure differences in their starting materials, nor differences in the
processing methods used, since all of the chocolates produced would have used similar,
but not identical, modern roasting and grinding techniques; the differences mainly
being the scale of the manufacture. Our results with unfermented beans clearly show
that there is a loss in antioxidant activity and flavanol content due to roasting
but that there is no significant difference between the traditional process and the
modern process in making chocolate mass.

Processing of Ivorian (fermented) beans

In Table 3 and in Figure 5 are presented the effects of traditional and modern processing on the flavanol content
of fermented Ivory Coast beans. Ivory Coast in 2009/10 represented the single largest
origin of cocoa in the world producing 1.242 tones of cocoa or 34.1% of total world
production [25]. The observations here are similar to the unfermented bean study. ORAC, epicatechin,
and flavanols DP 1–10 were significantly higher in traditional processed beans by
20% (p = 0.0021), 26% (p = 0.0075), and 22% (p = 0.0014) respectively. Conversely, catechin and total flavanols trended higher in
modern processed beans by 14% (p = 0.060) and 8% (p = 0.089) respectively, compared to traditionally processed beans.
There are two possible explanations for these results. First, cocoa bean fermentations
are done by very crude methods and the resulting fermented beans are highly heterogeneous
with respect to degree of fermentation. This may impact the level of starting antioxidant
capacity and flavanols in each bean. Therefore, one might expect more variability
in the data from fermented beans compared to data from unfermented beans. Second,
the higher terminal temperature in the modern processed beans partially, but does
not fully, explain the selective loss of only antioxidant activity, epicatechin and
flavanols DP 1–10 compared to the traditional process. But this does not explain why
catechin and total flavanols are numerically higher (not significant at p = 0.05) when processed by the modern method. If roasting had been done to the same
final temperature, it is likely that any differences might disappear [13]. Since these observations do not show a clear trend towards modern or traditional
processing, it can be concluded that there is little difference in the in vitro antioxidant activity and flavanol contents of the more fermented Ivory Coast beans
when processed in the traditional or modern way. This brings us to the same general
conclusion that the traditional and the modern processes for roasting and grinding
cocoa beans have about the same effect on the level of cocoa bean flavanols.

Figure 5.Comparison of fermented Ivory Coast (Ivorian) cacao beans that have been left unprocessed,
have been roasted and ground using traditional methods (Traditional Process) and have
been processed using modern processing. Top Graph. Analysis of antioxidant activity (ORAC); Second Graph. Analysis of epicatechin
(yellow bars) and catechin (blue bars) content; Third Graph, Analysis of Flavanols
DP 1–10 content and Bottom Graph, Analysis of Total Flavanol content.

Table 3.Antioxidant capacity, and flavanol content comparison for Ivory Coast beans roasted
on a Mayan-style comal and ground on a metate (Traditional process) compared to the
same beans roasted and ground using modern pilot plant equipment (Modern process)

When the Lavado bean processing data (Table 2) are compared to Ivory Coast processing data (Table 3), the same observation can be made that the antioxidant capacity and flavanol contents
of roasted, unfermented Lavado beans are significantly higher than in the roasted,
more-fermented Ivory Coast beans (Table 1). This also is consistent with data published by Payne, et al. [13] and others [10-12] where the flavanol monomer and oligomer content of roasted Lavado beans has been
found to be significantly higher than roasted beans from fermented sources like Ivory
Coast and Papua New Guinea.

Implications for traditional processing and modern processing

People native to Mesoamerica and Northern South America have a tradition of consuming
less-fermented or totally unfermented cocoa. This is due to the ease of processing
and drying cocoa beans in humid climates, particularly during the rainy season, when
the beans need to be quickly dried. Over the centuries these people have come to prefer
the unfermented, Lavado beans. Regardless of the processing methods used, Lavado beans
had the same level of flavanols and these levels were more than twice the level found
in fermented beans after similar processing (compare Table 2 to Table 3). This may mean that people native to the Americas today and before the discovery
of the New World by Columbus and Cortez, ate cocoa products made from cocoa mass (also
called chocolate liquor) that contained about twice the level of flavanols found in
today’s cocoa mass.

After Europeans began importing cocoa beans, first for use in beverages, and then
for solid chocolate, hot cocoa beverages and later milk chocolate, there was increased
demand for partially or fully fermented cocoa beans. The producers of cocoa in the
Americas were asked to produce cocoa beans that had been fermented to suit European
tastes and were paid a premium for these beans [3,4]. As is shown in this study (Table 1), and by others [9-13], fermentation lowers the level of flavanols in the raw beans. The benefit to the
European consumer was to have cocoa drinks or chocolate bars that were less bitter
and had more cacao flavor. With the increased use of fermented beans and with the
development of modern cocoa bean processing, there was a loss of flavanols from product,
with the greatest loss of flavanols due to the preference for fermented beans. The
results of the present study have shown that modern processing has little or no impact
on flavanols (Table 3, Figure 5). Thus the health benefits of cocoa and chocolate flavanols and other beneficial
bioactives need to be balanced with the flavor of the final product, for which fermentation
of cocoa beans tends to increase cacao flavor impact and lessen bitterness. Despite
the implementation of fermentation, dark chocolate and cocoa powder still have some
of the highest concentrations of flavanols of any food in our diet [26]. In fact a recent dose response study using conventionally processed cocoa powder
in ever increasing amounts has found that as little as 5 g is sufficient to elicit
significant vasodilation of the brachial artery, as measured by Flow Mediated Dilation
[27].

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

EC, KM and DS carried out cocoa roasting while MP performed and directed the analysis
of samples. WH assisted in experiment design, sample processing and directed the project.
All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank the Hershey Center for Health and Nutrition for providing
the samples and facilities for this research. We thank The Hershey Company for supplying
support to E. Chin as part of her undergraduate summer internship. Thanks go to John
Scharffenberger for his expert advice and direction for the ancient, traditional processing
of cacao beans. Also, a special thanks to W. J. Hurst for the acquisition of an authentic
clay comal from Latin America.